Molecular Characterization of Membranous Nephropathy: Quo Vadis?
Authors: Fernando C. Fervenza, Richard J. Glassock
Journal: Journal of the American Society of Nephrology (2022)

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. The lesion of MN is a "pattern of injury," not a specific diagnosis, which is based on finding bright granular IgG staining along the outer aspects of the glomerular basement membrane by immunofluorescence microscopy and subepithelial electron-dense deposits on electron microscopic examination. MN is believed to be immune mediated, involving in situ formation or deposition of antigen-antibody complexes as consequent to many etiologies. In the majority of cases, it presents without any immediately apparent underlying trigger, often categorized as primary MN. Patients with concomitantly recognized systemic diseases (e.g., autoimmune, malignancy, infections) or adverse drug reactions are traditionally categorized as secondary MN. After the discovery of PLA2R and THSD7A as target antigens in MN in 20091 and 2014,2 respectively, others have been identified.3,4 Each antigen represents a distinct form of MN, with different clinical and pathologic findings. Thus, it is now possible to redefine and subtype MN on the basis of its underlying molecular pathogenesis (e.g., PLA2R-, SEM3B-, NELL1-, PCDH7-, NCAM1-, etc. MN associated). These antigens account for approximately 90% of all forms of primary MN and can contribute to lesions formerly classified as secondary forms of MN. Thus, the concept of a discrete dichotomization of MN into primary and secondary forms has been substantially blurred and may no longer be relevant. Despite these transformative discoveries, our understanding of the triggering of the pathophysiologic processes that result in glomerular injury or its maintenance remain incomplete. The application of molecular transcriptomics to kidney biopsy tissue to characterize disease subtypes further has been proposed as an approach to "fine-tune" pathogenetic pathways; improve understanding of triggers, perpetuators, and prognostic factors; and develop patient-tailored therapies (precision medicine).5 In this issue of JASN, Sealfon et al.6 extended their earlier efforts by attempting to unravel the tissue molecular characteristics of the lesion of MN by applying state-of-the-art methodology with machine learning frameworks. These studies were designed to predict underlying pathogenesis by analyzing gene expression in microdissected kidney tissues from patients in the Nephrotic Syndrome Study Network (NEPTUNE)7 and the European Renal cDNA (ERCB) cohorts.8 They analyzed whole-genome glomerular microarrays from 125 patients (48 with MN) from NEPTUNE and 223 patients (21 with MN) from the ERCB cohort, which also included patients with FSGS, minimal change disease (MCD), and IgA nephropathy. An additional RNA sequence dataset in another 177 patients (20 with MN) from the NEPTUNE cohort served as a validation cohort. The researchers selected 500 genes assigned the highest feature importance by random forest classifier and performed hierarchical clustering analysis, which identified 158 MN-related genes. Patients with other lesions were not clearly separated. Next, a random forest classifier distinguished patients with each glomerular lesion, using whole-genome glomerular transcripts. A MN disease classifier performed better than all other lesion classifiers in both the NEPTUNE and ERCB cohorts. In contrast, using tubulointerstitial gene expression was unrevealing. The authors concluded that glomerular gene expression alone accurately separates participants with MN lesions from those with other nephrotic syndrome etiologies. Notably, genes encoding PLA2R, PCDH7, NELL1, and NCAM1 were among the differentially expressed genes detected. This intriguing finding deserves careful analysis. Due to the rarity of these MN lesion subtypes, it is unlikely that the NEPTUNE MN cohort included many patients with PCDH7- or NELL1-associated MN. Thus, several questions surface. First, is upregulation of these genes a broad effect unrelated to the specific antigen targeted in each case, i.e., PLA2R+ (the majority of patients with MN in the cohort)? Second, does generalized podocyte injury trigger expression of potential antigens in individuals with permissive genetic background? The 158 MN-related genes were further examined in a kidney-specific functional network.9 The genes clustered in modules involved with ion transport, cell projection morphogenesis, regulation of adhesion, and response to wounding. Data from single-cell RNA sequencing from reference nephrectomy tissues showed that 43% of these genes are mostly expressed by podocytes. The top-ranked classifier gene was FAM114A1, which encodes a poorly understood protein involved in neuronal development and possible interaction with the actin cytoskeleton. The authors claim that these MN-lesion specific genes did not correlate with age, proteinuria, and eGFR when analyses were restricted to 147 participants from the expanded NEPTUNE cohort with a urine protein creatinine ratio >3.5 (44 with MN). The authors also found the gene set to be significantly enriched in NFKB1 targets, which has been recently associated with increased risk of developing MN.10 Although the authors claim that their data provide a molecular snapshot of MN lesions that may facilitate insight into the underlying pathophysiology, much more is needed to validate such a claim, in our opinion. The reasons underlying this qualification are: First, it is important that the cohorts studied are clearly defined. It is not enough to say patients had FSGS, which is a lesion/"pattern of injury" found on histologic examination but is not a diagnosis. We are unsure how many patients had presumed primary, secondary, or genetic FSGS—a weakness the authors acknowledge. As such, validation of these findings utilizing properly characterized cohorts that include data on serum albumin, proteinuria, and electron microscopy analysis is needed. The same concerns could also apply to the MN lesion characterization used in the cohorts. Second, to evaluate fully the effect of the pathologic process, kidney biopsy should be evaluated at the time of presentation while there is clear active disease and before a patient is treated with immunosuppressive therapy. It is unclear how many biopsies in the entire cohort were from patients with active disease versus those in remission after immunosuppressive therapy. Third, the analyses of the influence of the magnitude of proteinuria (> or <3.5 g/d) upon the discriminatory (prediction accuracy) ability of the expression signature are problematic. As shown in Supplemental Figure 5, A–E, using area under the curve analysis, a high predictive accuracy (area under the curve=0.91) of the expression signature for a lesion of MN compared with FSGS or MCD was present in both the high and low proteinuria subgroups for the original NEPTUNE cohort.6 This finding was not reproduced in the expanded NEPTUNE cohort in comparison to MCD, especially in the low proteinuria MCD cohort. The ECRB cohort showed no difference in the predictive accuracy of the expression signature between MN and FSGS but did reveal good discrimination of MN from MCD. Insufficient numbers of patients with MCD were available in the ECRB cohort to make such comparisons in a low proteinuria group. In sum, it appears that the predictive accuracy of the expression signature is influenced by the magnitude of proteinuria, at least in some cohorts. This observation needs to be confirmed in larger and better characterized patient cohorts. Fourth, a deeper analysis of the overexpressed genes is needed to fulfill the authors' aim of providing pathophysiologic insights, to facilitate development of target therapeutics, and to improve tissue transcriptome-based disease classification. It seems clear that the "pattern-of-injury" lesion of MN is very heterogeneous, stemming from the remarkable progress in categorization of antigens operating in the pathogenesis of the lesion. Moving from this point to understanding of the significance of the underlying genetic architecture and transcriptomic signatures of MN will require much more work and collections of extremely well-characterized patients with a MN lesion to provide clinically meaningful information. Linking MN classifier genes to the pathophysiology of glomerular injury is a plausible path forward. If such linkage can be firmly established, then a good rationale and targets can be provided for the development of new therapeutics or biomarkers that could be truly relevant for the management of patients with a MN lesion or one of its many subtypes. The present paper should be welcomed as a beginning of the difficult research in this arena and a refreshing look at the challenges that lie ahead. Disclosures F. Fervenza reports consultancy for Alexion Pharmaceuticals, ByoCrystal, Galapagos, GSK, Novartis, Otsuka, and Takeda; research funding from Chemocentryx, Genentech, Hoffman La Roche, Janssen Pharmaceutical, Morphosys, and Retrophin; honoraria from UpToDate; and an advisory or leadership role for UpToDate, Kidney International, Nephrology, Nephrology Dialysis and Transplantation, and Journal of the American Society of Nephrology. R. Glassock reports consultancy for Agenx, Alexion, American Journal of Nephrology (Karger Publications), Anteris Bio, Arrowhead Bio, Aurinia, BioCryst, Calliditas, ChemoCentryx, Equillium, Forsee Pharma, Horizon, Ionis, the National Instituted of Health, Novartis, Omeros, Otsuka Pharma, RenaSight (Natera), River3Renal, Sentien, Therini Bio, Travere (Retrophin), UpToDate (Wolters-Kluwer), Vertex, Vivace, and Walden Bioscience; ownership interest in Reata, Inc.; honoraria from Agenx, Alexion, Anteris, Arrowhead, Aurinia, Biocryst, Chemocentryx, EcoR1, Horizon, Karger Publications, Natera, Novartis, Omeris, and Wolters-Kluwer (UpToDate); an advisory or leadership role for American Journal of Nephrology, BioCryst, Calliditas, JASN, Novartis, Otsuka, RenaSight, Travere, University Kidney Research Organization, and UpToDate; participated in a speakers' bureau for Aurinia; and has other interests or relationships with the American Society of Nephrology—open forum communities. Funding None.